PHOSPHORYLATED INTERMEDIATES IN THE SYNTHESIS OF SQUALENE

Abstract

Investigations in this laboratory on the mechanism of squalene synthesis from mevalonic acid have proceeded along two lines: (1) certain structural features of the "isoprenoid" condensing unit have been defined by experiments using heavy hydrogen as a tracer, and (2) yeast extracts have been fractionated with the aim of accumulating intermediates between MVA1 and squalene. The experiments with D202 and T-labeled MVA1 have led to the principal conclusions that the new carbon-carbon bonds between two isoprenoid units are formed by the interaction of two methylene groups and that the carboxyl groups of MVA are probably removed in a concerted process with simultaneous elimination of the tertiary hy-droxyl groups. As to the requirements for the overall process, it could be shown that in yeast extracts the co-factors for squalene synthesis from MVA are ATP, Mn++, and reduced pyridine nucleotide. To account for the requirements of ATP and manganese, the formation of phosphorylated intermediates was postulated .3 The subsequent demonstration of an enzyme system that catalyzes the reaction MVA + ATP-phospho-MVA + ADP confirmed this hypothesis.4 Phospho-MVA (P-MVA) is efficiently converted to squalene and appears to be the first product in the overall transformation. Evidence has been presented that P-MVA is a phosphate ester4 and, as will be shown later, can be assumed to be 5-phosphomevalonic acid. We have also reported briefly6 that ATP is still required for the further metabolism of P-MVA, suggesting the existence of additional phosphorylation steps. We now wish to describe a second and a third phosphorylated intermediate (II and III) which are formed during the conversion of MVA to squalene. EXPERIMENTAL